In aplastic anemia, the bone marrow is replaced by fat, and peripheral blood counts - - of white blood cells, red blood cells, and platelets - - fall to extremely low levels, leading to death from anemia, bleeding or infection. Aplastic anemia is a disease of young persons, and in its severe form is almost invariably fatal untreated. Historically, aplastic anemia has been linked to chemical exposures, in particular benzene; it is an idiosyncratic complication of some medical drug use; it occurs as a rare event in pregnancy and following seronegative hepatitis; and the diseases associated with certain immunologic conditions. The chance observation that some patients post-bone marrow transplant recovered their own marrow function led to the inference that the immunosuppressive conditioning regimen might have treated an underlying immune-mediated pathophysiology. Purposeful administration of antithymocyte globulin (ATG) has led to hematologic recovery in the majority of treated patients. Laboratory data have also revealed abnormalities of the immune system: lymphocyte populations that induce apoptosis in hematopoietic target cells by the Fas-mediated pathway, and oligoclones of effector T cells which express type 1 cytokines, especially gamma-interferon. The Hematology Branch has been a leader in both the scientific and medical studies of aplastic anemia pathophysiology and treatment. Three major clinical protocols have been completed with important implications for the care of patients with aplastic anemia. First, we have published our experience with CAMPATH, or alemutuzumab, a monoclonal antibody directed against the CD52 antigen on the lymphocyte cell surface and highly immunosuppressive. We employed alemutuzumab in several different protocols: as first line therapy for severe aplastic anemia, in patients refractory to first line treatment with ATG, and in patients who relapsed after successful ATG treatment. For relapse, CAMPATH was compared in a randomized trial to rabbit ATG; both second line therapies were approximately equally effective, inducing responses in 30-40% of patients. CAMPATH has the advantage of not requiring cyclosporine treatment, although more antibiotics prophylaxes are utilized. In relapsed disease, the majority of patients recovered hematopoietic function with either rabbit ATG or CAMPATH. Despite the clinical efficacy of CAMPATH in these settings, it was only minimally effective as first treatment in severe aplastic anemia, and this arm of a randomized three arm trial was terminated early. Second, we explored the utility of cyclophosphamide, a chemotherapy drug, at moderate doses, based on a Chinese report of efficacy that was comparable to ATG, with minimal toxicity. Cyclophosphamide at high doses has been advocated in aplastic anemia by others, but their results could not be reproduced in a randomized trial at NIH. Unfortunately, cyclophosphamide even at 60% of the high dose regimen, was extremely toxic, resulting in prolonged periods of neutropenia (in contrast to the Chinese report), fungal disease, and death in some patients. While the regimen was active, in that some patients did recover hematopoietic function, there were instances of both clonal evolution and relapse. The studies of moderate dose cyclophosphamide in both treatment-nave severe aplastic anemia and in refractory aplastic anemia were terminated early by the data and safety monitoring board, due to toxicity concerns. A particular worry was failure of several patients with adequate neutrophil counts to recover white cell levels, leading to prolonged hospitalizations and the requirement for high risk stem cell transplantation. Third, in a novel study, we utilized eltrombopag, a thrombopoietin minetic, in patients with refractory severe aplastic anemia. These patients have failed one or more courses with immunosuppression, as well as androgens and other growth factor trials in some cases. Thrombopoietin, and therefore eltrombopag, would be predicted to have effects at the level of the hematopoietic stem cell, from in vitro experiments, animal models, and observations of human patients with mutations in the receptor molecule. We were surprised at the benefit observed with eltrombopag, as 44% of the 25 patients who received eltrombopag showed unexpected hematologic improvement, most in more than one hematopoietic cell lineage. Increases in platelets and hemoglobin levels were substantial, leading to transfusion- independence. Neutrophil counts also rose. These results suggest that eltrombopag is active on the few remaining stem cells in aplastic anemia. Further studies are underway of eltrombopag in patients with refractory severe aplastic anemia, in moderate aplastic anemia, in myelodysplastic syndrome, and, for first treatment, eltrombopag in combination with horse ATG and cyclosporine to target both aberrant immune-mediated destruction of hematpoiesis with stimulation of hematopoietic stem cells early in the course of severe aplastic anemia. In tissue culture experiments, we have explored the finding in a recent report that fat cells (adipocytes) act as negative regulators of hematopoiesis in mice. We have utilized our immune mediated model of bone marrow failure, in which lymph node cells that are mismatched either for major H2 or minor H60 histocompatability loci are infused into recipients; recipients then develop aplastic anemia and pancytopenia 10-14 days later. The original observation of the role of adipogenesis in hematopoiesis was based on experiments employing a peroxisome proliferative activator receptor &#611; (PPAR-&#611;) inhibitor, as PPAR-&#611; is a key transcription factor in adipogenesis. We observed that PPAR-&#611; antagonists were active in mitigating immune-mediated marrow failure in our model. Animals receiving adequate doses of inhibitors showed more bone marrow cellularity and improved blood counts, including higher total numbers of Lin- Sca1+ c-kit+ stem cells. Confocal microscopy, as well as conventional staining of bone marrow showed striking differences between treated and untreated animals. These effects were dose responsive. However, we also noted in the PPAR-&#611; antagonist-treated animals significantly fewer CD8+ T cells, as determined by flow cytometry. We speculated that the antagonist might also affect the immune cells present in the donor inocculum. Indeed, we found the significant differences in concentrations of several inflammation-related cytokines in the plasma of animals, including interleukin-6 and TNF. PCR arrays for adipogenesis and inflammasome pathways showed that expression of adipogenesis genes was greatly decreased in treated groups, as expected, but inflammation and inflammasome related genes were also affected, while apoptosis inhibitor genes were upregulated. In vitro, the chemical inhibitors acted to suppress activation and proliferation of non-specifically stimulated T-cells. Therefore, the adipogenesis prohibitors appeared to be non- specific and to also affect T-cell activation. As confirmation, we found them inactive in ameliorating pancytopenia and bone marrow hypoplasia in animals subjected to irradiation or cytotoxic drug therapy.